Novel quinoline/thiazinan-4-one hybrids; design, synthesis, and molecular docking studies as potential anti-bacterial candidates against MRSA

In an attempt to develop effective and safe antibacterial agents, we synthesized novel thiazinanones by combining the quinolone scaffold and the 1,3-thiazinan-4-one group by reaction between ((4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)hydrazinecarbothioamides and 2,3-diphenylcycloprop-2-enone in refluxing ethanol in the presence of triethyl amine as a catalyst. The structure of the synthesized compounds was characterized by spectral data and elemental analysis, IR, MS, 1H and 13C NMR spectroscopy which showed two doublet signals for CH-5 and CH-6 and four sharp singlets for the protons of thiazinane NH, CH 
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Created by potrace 1.16, written by Peter Selinger 2001-2019
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 N, quinolone NH and OH, respectively. Also, the 13C NMR spectrum clearly showed the presence of two quaternary carbon atoms which were assigned to thiazinanone-C-5 and C-6. All the 1,3-thiazinan-4-one/quinolone hybrids were screened for antibacterial activity. Compounds 7a, 7e and 7g showed broad spectrum antibacterial activity against most of the tested strains either G +ve or G −ve. Compound 7e is the most potent antibacterial agent against MRSA with the minimum inhibitory concentration against MRSA found to be 48 μg mL−1 compared to the drug ciprofloxacin (96 μg mL−1). Additionally, a molecular docking study was performed to understand the molecular interaction and binding mode of the compounds on the active site of S. aureus Murb protein. In silico docking assisted data strongly correlated with the experimental approach of antibacterial activity against MRSA.

Large amounts of effort towards further research of quinolones are performed to develop new more effective antibacterial agents with broader antimicrobial spectrum and better therapeutic index. The azolylthioether quinolones III (Fig. 2) exhibited good antimicrobial activities that displayed remarkable anti-MRSA and anti-P. aeruginosa efficacies with low MIC values of 0.25 mg mL −1 , even superior to reference drugs. They induced bacterial resistance more slowly than clinical drugs. 35 Also, compound IV (Fig. 2), 3-aminothiazolquinolones, 3-(2-aminothiazol-4-yl)-7-chloro-6-(pyrrolidin-1-yl)quinolone exhibited potent antibacterial activity, low cytotoxicity to hepatocyte cells, strong inhibitory potency to DNA gyrase and a broad antimicrobial spectrum including against multidrug-resistant strains. This active molecule IV also induced bacterial resistance more slowly than noroxacin. 36 Moreover, thiazinane was taken into consideration in MDR challenge whereas the monoiodinated thiazine derivative V (Fig. 2) showed good antibacterial activity against methicillin-sensitive Staphylococcus aureus (S. aureus, MSSA) ATCC 29213 and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. Among strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sublethal concentration of 3-phenyl-1,4-benzothiazine VI (Fig. 2), at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine. 37 In response to the previously mentioned ndings, we here designed novel compounds based on the concept of merging more than one scaffold in one compact structure. Hybrids 7a-h gather two types of anti-MRSA scaffolds; 2-quinolones and 1,3thiazines in one novel hybrid aims to develop simpler and more efficient antibacterial compounds with synergistic effect and less bacterial resistance. Testing against G +ve and G −ve bacteria align with examining against methicillin-resistant S. aureus (MRSA) to investigate the anti-MDR activity as well as anti-bacterial spectrum. This is illustrated in a summarized schematic diagram (Fig. 2).
Diastereomeric mixtures 7a-h were formed as a result of the development of two new stereo centers at C-5 and C-6 positions. As a result, most signals in the 1 H and 13 C NMR spectra were duplicated. The expected diastereomeric forms were not separable by column chromatography. In the 1 H NMR spectra, the signals of the respective protons of the synthesized compounds were conrmed based on their chemical shis and multiplicities. The compounds reported in this study have been thoroughly characterized by elemental analysis and mass spectral data. The 1 H NMR spectrum of 7a showed, in addition to the aromatic protons, two doublet signals for CH-6 and CH-5 at d H = 4.50 ppm and 5.44 ppm with coupling constant J = 4.0 Hz and four sharp singlets at d H = 8.15, 8.55, 11.60 and 12.03 ppm related to the protons of thiazinane NH, CH]N, quinolone NH and OH, respectively. As the saturated thiazinanes belong to the cyclohexane conrmation structure, the coupling constant values of CH-5 and CH-6 = 4 Hz. Moreover, the 13 C NMR spectrum clearly showed the presence of two quaternary carbon atom which resonated at d C = 43.36 and 56.06 ppm which were assigned to thiazinanone-CH-6,5. Furthermore, the 13  According to elemental analysis and mass spectrometry, compound 7a has a molecular formula of C 26 H 20 N 4 O 3 S, resulting from the addition of one molecule of hydrazinecarbothioamide 5a with one molecule of 6 without any elimination.
In case of 7b, its 1 H NMR spectrum showed triplet and quartet signals for CH 3  The plausible mechanism for the formation of 1,3-thiazinan-4-ones 7a-h was based upon the conjugate double bond of 6 was attacked by the thione lone pair forming zwitterion salts 8a-h. Subsequently, a proton was transferred in 8a-h to give the intermediates 9a-h, which on rearrangement and ring opening of cyclopropenone would give the intermediate 10a-h. The carbonyl carbon was then attacked by the lone-pair of nitrogen to form intermediates 11a-h, which rearranged to give the nal products 7a-h (Scheme 2).  Using compound 7a as an example, we carried out the reaction in various settings aer optimizing the reaction conditions. When the reaction was reuxed in DMF/Et 3 N and dioxane/Et 3 N, it was found that the yield of 7a was reduced to 60% and 64%, respectively. In addition, side products were obtained from the reaction. As a result, utilizing ethanol in the presence of Et 3 N as a catalyst is the best way to get high yields.

Screening of antibacterial activity
The antibacterial and antifungal activities of compounds 7a-h were evaluated in vitro against three-Gram positive (G +ve) strains; non-resistant S. aureus (ATCC 6538), methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli (ATCC 25922) and two-Gram negative (G −ve) strains; Pseudomonas aeruginosa (ATCC 10145) and Salmonella. The tested compounds were assayed against ciprooxacin as an antibacterial reference using standard agar cup diffusion method. Results of the antibacterial screening are listed in Table 1.
According to the MICs recorded in Table 1, it can be deduced that most of the tested compounds showed a higher antibacterial activity than the reference ciprooxacin against G +ve bacteria. It was found that compound 7a, 7e and 7h displayed potent activity against non-resistant S. aureus compared with the reference with MICs of 12, 48, and 48 mM, respectively. Meanwhile, compounds 7e displayed signicant antibacterial activity against MRSA better than the reference with MICs 48 and 96 mM, respectively, however 7a showed remarkable activity of MIC 96 mM.
Moreover, compounds 7g exhibited the good activity against E. coli with MICs of 24 mM when compared to the reference, however, both 7a and 7d showed moderate activity against E. coli.
Concerning activity against G −ve strains; compounds 7e and 7g revealed a high potency against P. aeruginosa with MICs of 12 and 12 mM. On contrast, other compounds displayed moderate to weak activity (Fig. 4). Furthermore, the derivatives 7d-f showed moderate activity against Salmonella with MICs of 96 mg mL −1 , respectively (Table 1 and Fig. 4).
2.2.1. Structure-activity relationship. Based on the aforementioned results, it is obvious that compounds 7a, 7e and 7g showed broad spectrum antibacterial activity against all the tested strains either G +ve or G −ve. In general, the quinolonebased thiazine derivatives 7c and 7f exhibited weak activity almost against most of the tested strains. From the above results, it can be concluded that, there is no specic substituent on the quinolone nucleus of tested compounds to enhance the antibacterial activity in a broad-spectrum manner. So, the enhanced activity of some of the tested derivatives may be due to improvement of the physicochemical properties and consequently enhancing permeability to microbial cells.
In summary, compound 7e presented a signicant broad spectrum anti-bacterial activity that was probably attributed to when (R 1 = CH 3 ), it would enhance the physicochemical parameters and hence increase cell permeability against either nonresistant or resistant strains.

Molecular modeling studies
Docking studies have been carried out to elucidate the binding mode of the quinolone/thiazine hybrids 7a-h with the protein active site of S. aureus Murb (PDB ID: IHSK). Prior to the molecular docking studies, the receptor protein was prepared for docking by omitting additional water and co-factors, followed by the addition of polar hydrogens and computing charges xation. Also, the docking scores of the tested compounds are depicted in Table 2 that used to calculate the inhibition constant (K i value) according to the reported equation 7 (see ESI †). Typically, a high potency is implied by a low K i value and it has to be in the micromolar range for a molecule to be qualied as a lead compound or hit. Compounds 7b, 7c, 7e and 7h have the least K i value of 0.62 × 10 −6 , 0.83 × 10 −6 , 0.78 × 10 −6 and 0.76 × 10 −6 mM, respectively to qualify as a drug and hence, the most potent among the other tested compounds.
Docking results of the known antibacterial reference; cipro-oxacin into active site of S. aureus Murb protein ( Fig. 5 and Table 2) revealed that ciprooxacin showed CDOCKER energy of −6.53 kcal mol −1 and engaged in two hydrogen bonds with amino acid residues SER82 and GLY79.
Most of the tested compounds have high binding affinity to protein of S. aureus Murb as the binding free energy (DG) values of them range from 0.0 to −2.6 kcal mol −1 . The docking study results of target 7b, 7c and 7f showed interactions typically as the reference with amino acid residues SER82 and GLY79.
Although, all the tested compounds 7a-h showed interaction with amino acid residues SER82 and TYR149, hybrids 7a, 7e and 7h lack interaction with the last residue.
Moreover, compounds 7e and 7h exhibited potential interactions with both amino acid residues ASN80 and ARG255, while the hybrid 7d interacted with the rst residue and the second residue engaged with the 7a.
Interestingly, compound 7f showed additional two hydrophobic interactions with ILE140 amino acid residue which is not observed with the others.
Collectively, the docking results were in agreement with the biological study, and we could conclude that hybrid 7e entitled to be promising as attractive future lead candidate for the development of broad-spectrum antibacterial activity.
3.1.2. General procedure. Equimolar amounts of 2,3diphenylcycloprop-2-enone 6 and the appropriate hydrazinecarbothioamides 5a-e were mixed in absolute EtOH and a few drops of Et 3 N was added as a catalyst and reuxed for about 4-6 h, furnished yellow precipitates (i.e. the reaction was followed up by TLC analysis). The precipitate was ltered, washed with ethanol, dried and recrystallized from the stated solvents to give the nal products 7a-h.

Biology
3.2.1. Screening of antibacterial activity. The antibacterial activity was screened according to serial dilution method. Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism aer overnight incubation (see ESI †).
3.2.2. Molecular docking study. The docking simulation study was carried out using Molecular Operating Environment (MOE®) version 2014.09 (Chemical Computing Group Inc., Montreal, QC, Canada). The computational soware operated under "Windows XP" installed on an Intel Pentium IV PC with a 1.6 GHz processor and 512 MB memory. The target compounds were constructed into a 3D model using the builder interface of the MOE program and docked into the active site of caspase-3 (PDB: 3GJQ). Checking their structures and the formal charges on atoms by 2D depiction was carried out and the energy, was minimized until an RMSD (root mean square deviations) gradient of 0.01 kcal mol −1 and RMS (Root Mean Square) distance of 0.1 A with MMFF94X (Merck molecular force eld 94X) force-eld and the partial charges were automatically calculated (see ESI †).

Conclusions
In short, a series of 1,3-thiazinanone derivatives have been synthesized in excellent yields via nucleophilic attack of thiosemicarbazones on 2,3-diphenylcyclopropenone. The target compounds were identied and characterized using 1 H NMR, 13 C NMR, MS and elemental analysis. The suggested mechanism for the formation of the nal products was remembered. The biological results revealed that some target compounds exhibited good antibacterial activity against most of the tested G +ve and G −ve strains, especially compound 7e against MRSA even superior to reference drug. They induced bacterial resistance more slowly than clinical drugs. Molecular docking study indicated strong binding interaction of the tested compounds.